1. Field of the Invention
The present invention relates to the field of medicine. More specifically, it relates to blood and blood products, and uses of those products to identify diseases and disorders affecting the blood or components of it. In many instances, the present invention relates to the field of dry platelet preparations and their use in diagnosis and monitoring of diseases and disorders relating to platelet function.
2. Description of Related Art
Platelets are formed in the bone marrow as fragments of megakaryocytes. They are irregularly-shaped, colorless bodies that are present in blood at a concentration of 150,000-450,000 per microliter (ul). Platelets play a crucial role in normal hemostasis, and they are the first line of defense against blood escaping from injured blood vessels. When bleeding from a wound suddenly occurs, the platelets gather at the wound and attempt to block the blood flow by forming a clot. The sticky platelets adhere to the damaged area and gradually form a platelet plug. At the same time, the platelets release a series of chemical signals that prompt other factors in the blood to reinforce the platelet plug. Between the platelet and its reinforcements, a sturdy clot is created that acts as a patch while the damaged area heals.
Blood clotting is a complicated process: if the clot formation is unchecked, the vessel will become occluded; if the clot is not sturdy, excessive blood loss will occur. Therefore, a delicate balance must be maintained for normal hemostasis. In situations where normal hemostasis is unbalanced, clot formation may be compromised. Such an abnormality could be acquired due to ingestion of aspirin or caused by immune dysfunction. The abnormality could also be congenital, such as through genetic diseases and clotting factor defects. For example, defects in the process of hemostasis that lead to bleeding disorders have been identified, and most of such defects are in the enzymes involved in the cascade of activities required for clotting, in platelet activation and function, or in contact activation. Included among these disorders are von Willebrand's Disease (vWD) and hemophilia. Other diseases or disorders of the blood clotting system are a result (i.e., side effect) of treatments for other diseases or disorders. Treatments for such diseases and disorders typically involve reducing the dose of the drug causing the side effect, or discontinuing treatment with the drug.
Typically, detection of a blood clotting disease or disorder involves analyzing the patient's blood for platelet counts, various markers involved in blood clotting, and clot-forming ability. The coagulation assays measuring the activated clotting time (ACT), the prothrombin time (PT), the plasma thrombin time (PTT), and the activated partial thromboplatin time (APTT) are used to evaluate the intrinsic and extrinsic coagulation pathways. These assays are generally performed in the laboratory and analysis often requires multiple samples of blood to be drawn from the patients. Moreover, these assays are potentially unreliable as they are end-point tests in which results are based on the time of clot formation in vitro. Another limitation relates to the fact that exogenous reagents, such as kaolin, thrombin, calcium, etc. must be added; thus, the results are based on an artificial system, and do not necessarily reflect the patient's thrombotic potential.
Platelet functionality is another critical component of blood clots. Dysfunctional platelets can lead to abnormal hemorrhage, such as bleeding or thrombosis. Thus, platelet function assays are an integral part of the diagnosis and monitoring of blood related diseases. For example, acquired platelet defects, such as ingestion of aspirin, cardiac disease, renal disease, or congenital platelet defects such as Bernard-Soulier syndrome, Glanzmann's thrombasthenia and storage pool disease, to name a few, can influence the normal hemostatic function of the platelets. To assess the platelet function, at the very minimal, a complete blood count with a peripheral blood smear will provide some basic information. Other tests, such as bleeding time, platelet function tests using an aggregometer to assess the aggregation of platelets to a panel of platelet agonists performed on whole blood or platelet rich plasma, will classify the defect. However, such analyses, although accurate, are not highly sensitive, and can fail to detect slight perturbations in normal clotting function at early stages of a disorder. Likewise, determination of the precise point of failure of the blood clotting cascade may require numerous assays using freshly drawn blood.
One proposal for use of platelets as hemostatic agents and in diagnostics is to prepare platelets in a freeze-dried state and store them for later use. Many groups have attempted to produce suitable freeze-dried platelets for various uses. For example, U.S. Pat. No. 5,622,867 to Livesey et al. discloses a system for cryoprotecting platelets for storage. The system treats fresh platelets with an inhibitor system comprising second messenger effectors. Inhibitors of one or more of the following pathways are added: cAMP, sodium channel, cGMP, cyclooxygenase, lipoxygenase, phospholipase, calcium, proteinase and proteinase, and membrane modification. A cryoprotectant, such as dimethyl sulfoxide (DMSO), maltodextrin, dextran, hydroxyethyl starch, and glucose, is also added where the platelets are to be maintained at low temperatures. Prior to use, the platelets are washed to remove the inhibitors and cryoprotectant.
In addition, U.S. Pat. No. 5,656,498 to Iijima et al. discloses freeze-dried platelets and methods of making them. The method comprises pre-treating platelets in blood plasma with a solution containing a saccharide, a biopolymer, an acid, or an acid salt, granulating the treated plasma, rapidly cooling the granules, and freeze-drying the granules. Furthermore, U.S. Pat. No. 5,736,313 to Spargo et al. discloses freeze-dried platelets and a process for making them. The process of making the freeze-dried platelets according to that patent comprises pre-incubating the platelets in a phosphate-citrate buffer or a phosphate-phosphate-citrate buffer, both of which contain a carbohydrate (e.g., glucose). After pre-incubation, the platelets are loaded with a carbohydrate, then suspended in a lyophilization buffer containing a matrix-forming polymer and a carbohydrate. The platelets are then slowly cooled to about −50° C. while the pressure is reduced to a vacuum state.
U.S. Pat. Nos. 5,958,670 and 5,800,978, both to Goodrich et al., also disclose freeze-dried platelets and methods of making them. The inventions disclosed in these patents rely on use of compositions having glass transition temperatures of above about −60° C. The compositions generally comprise a component that is permeable to the platelets (e.g., a carbohydrate, such as a sugar) and a component that is impermeable to the platelets (e.g., gelatin, PEG). To create the freeze-dried platelets, the temperature of the composition is reduced to a point below the glass transition temperature of the composition, and vacuum evaporating or subliming the liquid from the composition. An earlier patent, U.S. Pat. No. 5,213,814, also to Goodrich et al., discloses stabilized platelets and methods of making them. The methods and platelets are suitable for storage of the platelets for extended periods of time at about 4° C. The methods generally comprise immersing platelets in a buffered aqueous solution containing a carbohydrate and a biologically compatible polymer or mixture of polymers, then freezing the solution and drying the frozen solution to produce freeze-dried platelets containing less than 10% by weight of moisture.
U.S. Pat. Nos. 6,127,111 and 6,372,423, both to Braun, disclose freeze-dried platelets and methods of making them. The methods of making the freeze-dried platelets comprise exposing the platelets to a coagulation inhibitor (e.g., EDTA or citrate) and a “cake forming agent” (e.g., a protein such as serum albumin, or a polysaccharide such as mannitol) for about 5 to 60 minutes at room temperature, and then freeze-drying to reduce the moisture content to below 10%.
Investigators at the University of California, Davis, have developed a process for making freeze-dried platelets which comprises loading the platelets with trehalose prior to freeze-drying. In U.S. Pat. No. 6,723,497, a method of preparing freeze-dried platelets is disclosed in which platelets are loaded with trehalose by incubating the platelets at a temperature from about 25° C. to less than about 40° C. with up to 50 mM trehalose, cooling the loaded platelets to below −32° C., and lyophilizing the cooled platelets. Published U.S. patent application 2005/0048460 discloses a method for making freeze-dried platelets that includes exposing the platelets to a carbohydrate (e.g., trehalose) and an amphiphilic agent (e.g., arbutin), and freeze-drying the platelets. Other patents and patent application publications relating to this field include, for example, U.S. Pat. Nos. 6,770,478, 6,723,497, 5,827,741, and U.S. published patent applications numbers 2005/0048460, 2004/0152964, 2004/0147024, and 2004/0136974.
U.S. Pat. No. 6,833,236 to Stienstra discloses a method for the production of stabilized platelets, and platelets made by the method. The method comprises pre-activating the platelets, for example by exposing them to stress, to induce formation of microvesicles, contacting the pre-activated platelets with a carbohydrate to introduce the carbohydrate into the platelets, and drying the loaded platelets.
The inventors and their colleagues have likewise developed freeze-dried platelets, compositions containing them, and methods of making them. Their inventions are disclosed, for example, in U.S. patent application Ser. Nos. 11/197,310 and 11/152,774, and in international patent application PCT/US2005/028559. An exemplary method disclosed in one or more of these patent documents includes loading platelets with 100 mM trehalose in the presence of 1% ethanol. The method also includes addition of 6% (final concentration) of polysucrose 400 as a stabilizing matrix before lyophilization.
Although numerous groups have developed processes for making freeze-dried platelets, and even though numerous advances in detecting and treating bleeding disorders have taken place over the last several years, the inventors have recognized that there is still a need for improved compositions and methods for detecting such disorders, particularly more sensitive and accurate methods to detect the development of the disorders.